Phenotypic and functional characterization of aqueous humor derived extracellular vesicles.

Extracellular vesicles (EVs) are double membrane vesicles, abundant in all biological fluids. However, the characterization of EVs in aqueous humor (AH) is still limited. The aim of the present work was to characterize EVs isolated from AH (AH-EVs) in terms of surface markers of cellular origin and functional properties. We obtained AHs from patients with cataract undergoing surgical phacoemulsification and insertion of intraocular lenses (n = 10). Nanoparticle tracking analysis, electron microscopy, super resolution microscopy and bead-based cytofluorimetry were used to characterize EVs from AH. Subsequently, we investigated the effects of AH-EVs on viability, proliferation and wound healing of human immortalized keratinocyte (HaCaT) cells in vitro in comparison with the effect of mesenchymal stromal cell-EVs (MSC-EVs). AH-EVs had a mean size of around 100 nm and expressed the classical tetraspanins (CD9, CD63 and CD81). Super resolution microscopy revealed co-expression of CD9, CD63 and CD81. Moreover, cytofluorimetric analysis highlighted the expression of mesenchymal, stem, epithelial and endothelial markers. In the in vitro wound healing assay on HaCaT cells, AH-EVs induced a significantly faster wound repair, comparable to the effects of MSC-EVs, and promoted HaCaT cell viability and proliferation. We provide evidence, herein, of the possible AH-EV origin from stromal cells, limbal epithelial/stem cells, ciliary epithelium and corneal endothelium. In addition, we showed their in vitro proliferative and regenerative capacities.

MicroRNA-22 Is a Key Regulator of Lipid and Metabolic Homeostasis.

Obesity is a growing public health problem associated with increased risk of type 2 diabetes, cardiovascular disease, nonalcoholic fatty liver disease (NAFLD) and cancer. Here, we identify microRNA-22 (miR-22) as an essential rheostat involved in the control of lipid and energy homeostasis as well as the onset and maintenance of obesity. We demonstrate through knockout and transgenic mouse models that miR-22 loss-of-function protects against obesity and hepatic steatosis, while its overexpression promotes both phenotypes even when mice are fed a regular chow diet. Mechanistically, we show that miR-22 controls multiple pathways related to lipid biogenesis and differentiation. Importantly, genetic ablation of miR-22 favors metabolic rewiring towards higher energy expenditure and browning of white adipose tissue, suggesting that modulation of miR-22 could represent a viable therapeutic strategy for treatment of obesity and other metabolic disorders.

Routes of Stem Cell Administration.

Stem cells are very promising for the treatment of a plethora of human diseases. Numerous clinical studies have been conducted to assess the safety and efficacy of various stem cell types. Factors that ensure successful therapeutic outcomes in patients are cell-based parameters such as source, viability, and number, as well as frequency and timing of intervention and disease stage. Stem cell administration routes should be appropriately chosen as these can affect homing and engraftment of the cells and hence reduce therapeutic effects, or compromise safety, resulting in serious adverse events. In this chapter, we will describe the use of stem cells in organ repair and regeneration, in particular, the liver and the available routes of cell delivery in the clinic for end-stage liver diseases. Factors affecting homing and engraftment of stem cells for each administration route will be discussed.

Long-term follow-up of diabetic and non-diabetic patients with chronic hepatitis C successfully treated with direct-acting antiviral agents.

BACKGROUND AND AIMS: Clearance of hepatitis C virus (HCV) is associated with improved glycometabolic control in patients with diabetes mellitus (DM) but whether this effect is maintained over the long term with a reduction in liver-related events (LRE) is still debated. To address these issues, we conducted a long-term prospective study on diabetic and non-diabetic patients with chronic hepatitis C cured by direct antiviral agents (DAAs). METHODS: Among 893 recruited patients, 15.7% were diabetic (Group 1) and 84.3% non-diabetic (Group 2); changes in fasting glucose (FG) and glycated haemoglobin (HbA1c) levels were assessed in Group 1 while the incidence of LRE was established in the whole cohort. Differences between groups were evaluated and independent predictors of unfavourable clinical outcome were established. RESULTS: After a mean follow up of 44.5 months, a significant reduction in FG and HbA1c values was found in Group 1. Death was reported in 5.7% of patients in Group 1 vs 1.6% in Group 2 (P = .003), hepatocellular carcinoma (HCC)-free survival was significantly lower in Group 2 (P = .015) as well as LRE-free survival in Group 1 cirrhotic patients (P = .0006). After adjustment for baseline variables, cirrhosis and albumin levels emerged as independent predictors of LRE; low albumin levels, DM and central obesity were associated with HCC risk in cirrhotic patients while insulin therapy emerged as unfavourable predictor among diabetics. CONCLUSIONS: SVR achieved by DAAs is associated with long-term improvement of glycometabolic control in diabetic patients, but among cirrhotics DM still exerts a detrimental effect on the liver.

Review: Extragastric diseases and Helicobacter pylori.

The involvement of Helicobacter pylori infection in many extra-gastroduodenal manifestations remains a fascinating field of investigation. However, for several of these supposed associations, the potential pathogenic mechanism remains unclear. The present review highlights the main associations of H pylori with extra-gastroduodenal manifestations reported during the last year. We searched for the most relevant studies on this topic, published between April 2019 and March 2020, identified using the term "Helicobacter" in the MEDLINE/Pubmed database. Consistent data emerged from studies investigating metabolic syndrome and ischaemic cardiovascular diseases. Other reported fields of investigation were hepatology, especially focused on non-alcoholic steatohepatitis, neurology, including Parkinson's disease and Alzheimer's disease, as well as dermatology. Inflammatory bowel disease (IBD), that comprises Crohn's disease and ulcerative colitis, may originate from a dysregulation of the host's immune response to commensal bacteria in individuals with genetic predisposition. The reduction of biodiversity and other specific imbalances in the faecal microbiome composition of IBD patients compared to that of healthy controls support this hypothesis. In this context, an inverse correlation between H pylori infection and IBD prevalence has been confirmed. Similar results were found in patients with kidney diseases and allergic manifestations. There are indications of the possible involvement of H pylori infection in metabolic syndrome and ischaemic cardiovascular diseases. However, due to a series of factors linked to study designs and the multifactorial pathogenesis of some diseases, further studies are needed.

Proteomics-Based Evidence for a Pro-Oncogenic Role of ESRP1 in Human Colorectal Cancer Cells.

The RNA-binding protein, Epithelial Splicing Regulatory Protein 1 (ESRP1) can promote or suppress tumorigenesis depending on the cell type and disease context. In colorectal cancer, we have previously shown that aberrantly high ESRP1 expression can drive tumor progression. In order to unveil the mechanisms by which ESRP1 can modulate cancer traits, we searched for proteins affected by modulation of Esrp1 in two human colorectal cancer cell lines, HCA24 and COLO320DM, by proteomics analysis. Proteins hosted by endogenous ESRP1 ribonucleoprotein complex in HCA24 cells were also analyzed following RNA-immunoprecipitation. Proteomics data were complemented with bioinformatics approach to exploit publicly available data on protein-protein interaction (PPI). Gene Ontology was analysed to identify a common molecular signature possibly explaining the pro-tumorigenic role of ESRP1. Interestingly, proteins identified herein support a role for ESRP1 in response to external stimulus, regulation of cell cycle and hypoxia. Our data provide further insights into factors affected by and entwined with ESRP1 in colorectal cancer.

Incidence of Prostate Cancer in Inflammatory Bowel Disease: A Meta-Analysis.

Background and objectives: Inflammatory bowel disease (IBD) is associated with an increased risk of developing colorectal cancer as well as some extra-intestinal tumors, but there are still limited data about the risk of prostate cancer (PC). To analyze if there is an increased risk of PC in patients affected by IBD, we performed a systematic review with meta-analysis. Materials and Methods: A Pubmed search of all studies comparing standardized incidence ratio (SIR) or odds ratio (OR) or relative risks (RR) of PC between IBD and non IBD groups, published until March 2020 was conducted. The study protocol was registered on PROSPERO. Twelve studies, mostly population studies, were included. The quality score of these studies, evaluated by the Newcastle-Ottawa Scale, was 7. The heterogeneity was high among the studies in which ulcerative colitis (UC) was considered separate from Crohn's disease (CD) and in the studies that considered UC and CD together ("IBD-studies"), while it was low in the studies which considered CD separate from UC. Results: The relative risk of developing PC was 1.71 (95% confidence interval [CI] 1.16-2.51, p = 0.007) in IBD, 1.10 (95%CI 0.98-1.25, p = 0.116) in CD, and 1.22 (95%CI 0.98-1.51, p = 0.07) in UC. Conclusions: Patients with IBD appear to have a slightly increased risk of PC compared to the general population.

Comments on Umer Draz et al. (J Pak Med Assoc. 2018; 68: 939-41) Presence of pre-diabetes in Helicobacter pylori positive versus Helicobacter pylori negative patients having dyspepsia.

We have read with great interest the article by Draz et al, reporting the prevalence of Helicobacter pylori (H. pylori) infection, in patients with dyspepsia and pre-diabetic status. The authors found that 58.5% of patients with H. pylori infection versus 45.9% (p=0.03) of patients without H. pylori had pre-diabetes. The method used to diagnose H. pylori infection was the histology based on bioptic samples taken in antrum during gastroscopy.1 This latter point raises a crucial criticism. When a patient needs a gastroscopy, due to alarming symptoms or older age (generally >45 years, but age should be determined locally according to gastric cancer risk), H. pylori infection can be detected on biopsies taken in the stomach from two topographical locations, the antrum and the corpus. Since the inherent risk of any biopsy procedure is sampling error, to biopsy both gastric compartments (two biopsies from the antrum and two from the corpus) increases the test's sensitivity, especially if the patient was recently treated with a proton pump inhibitor drug.2 When gastroscopy is not mandatory, C-urea breath test (UBT) is the most accurate tool to detect H. pylori infection and is more cost-effective than endoscopy. This method is based on the principle that, in the presence of H. pylori urease, labelled carbon dioxide is exhaled in the expired breath. In contrast to biopsy-based tests, UBT is not liable to sampling errors (it tests the entire stomach).2 Thus, although the findings of Draz et al agree with a large series of studies,3 the accuracy of their findings could be increased avoiding the risk of false-negative results due to the limited area of stomach investigated for H. pylori infection.

A composite enhancer regulates p63 gene expression in epidermal morphogenesis and in keratinocyte differentiation by multiple mechanisms.

p63 is a crucial regulator of epidermal development, but its transcriptional control has remained elusive. Here, we report the identification of a long-range enhancer (p63LRE) that is composed of two evolutionary conserved modules (C38 and C40), acting in concert to control tissue- and layer-specific expression of the p63 gene. Both modules are in an open and active chromatin state in human and mouse keratinocytes and in embryonic epidermis, and are strongly bound by p63. p63LRE activity is dependent on p63 expression in embryonic skin, and also in the commitment of human induced pluripotent stem cells toward an epithelial cell fate. A search for other transcription factors involved in p63LRE regulation revealed that the CAAT enhancer binding proteins Cebpa and Cebpb and the POU domain-containing protein Pou3f1 repress p63 expression during keratinocyte differentiation by binding the p63LRE enhancer. Collectively, our data indicate that p63LRE is composed of additive and partly redundant enhancer modules that act to direct robust p63 expression selectively in the basal layer of the epidermis.

Prevalence and prognostic value of hepatic histological alterations in patients with Crohn's disease.

UNLABELLED: Variable degrees of liver histological changes in patients with Crohn's disease (CD) have been reported. OBJECTIVE: To evaluate the liver histological alterations and their prognostic significance in patients affected by CD without abnormalities of liver biochemical parameters and ultrasound features. MATERIAL AND METHODS: A prospective, single-blind study, including 35 consecutive patients with CD that underwent bowel resection with a contemporary performance of liver biopsy from 1992 to 2003. EXCLUSION CRITERIA: the presence of standard causes of liver disease, such as alcohol consumption exceeding 20 g/day, primary sclerosing cholangitis, viral infections, celiac disease, metabolic syndrome and alterations of the metabolism. Patients were followed up with regular evaluation of hepatic cytolysis, cholestasis, synthesis and ultrasound performance. After a mean interval of 14 years (from May to December 2013), liver fibrosis was assessed by Fibroscan®. RESULTS: Histological alterations were shown in 60% of patients, without serious liver injuries (no case of inflammation or significant fibrosis). Fibroscan® was performed in 33 subjects and no significant changes were observed (mean value of liver stiffness: 5.2 ± 1.2 kPa). The minimal microscopic damage did not evolve either in patients with a normal histology or in those with an altered histology at baseline (p = 0.9). Only patients who took azathioprine had a statistically significant increase of liver stiffness values (5.7 ± 1.5 kPa vs 4.7 ± 1.3 kPa, p = 0.017). CONCLUSIONS: Patients with CD do not need additional examinations compared to the general population, unless clinical or biochemical abnormalities are found.

Renal cells from spermatogonial germline stem cells protect against kidney injury.

Spermatogonial stem cells reside in specific niches within seminiferous tubules and continuously generate differentiating daughter cells for production of spermatozoa. Although spermatogonial stem cells are unipotent, these cells are able to spontaneously convert to germline cell-derived pluripotent stem cells (GPSCs) in vitro. GPSCs have many properties of embryonic stem cells and are highly plastic, but their therapeutic potential in tissue regeneration has not been fully explored. Using a novel renal epithelial differentiation protocol, we obtained GPSC-derived tubular-like cells (GTCs) that were functional in vitro, as demonstrated through transepithelial electrical resistance analysis. In mice, GTCs injected after ischemic renal injury homed to the renal parenchyma, and GTC-treated mice showed reduced renal oxidative stress, tubular apoptosis, and cortical damage and upregulated tubular expression of the antioxidant enzyme hemeoxygenase-1. Six weeks after ischemic injury, kidneys of GTC-treated mice had less fibrosis and inflammatory infiltrate than kidneys of vehicle-treated mice. In conclusion, we show that GPSCs can be differentiated into functionally active renal tubular-like cells that therapeutically prevent chronic ischemic damage in vivo, introducing the potential utility of GPSCs in regenerative cell therapy.

RNA-binding protein transcripts as potential biomarkers for detecting Primary Sclerosing Cholangitis and for predicting its progression to Cholangiocarcinoma.

Primary Sclerosing Cholangitis (PSC) is a persistent inflammatory liver condition that affects the bile ducts and is commonly diagnosed in young individuals. Despite efforts to incorporate various clinical, biochemical and molecular parameters for diagnosing PSC, it remains challenging, and no biomarkers characteristic of the disease have been identified hitherto. PSC is linked with an uncertain prognosis, and there is a pressing need to explore multiomics databases to establish a new biomarker panel for the early detection of PSC's gradual progression into Cholangiocarcinoma (CCA) and for the development of effective therapeutic interventions. Apart from non-coding RNAs, other components of the Ribonucleoprotein (RNP) complex, such as RNA-Binding Proteins (RBPs), also hold great promise as biomarkers due to their versatile expression in pathological conditions. In the present review, an update on the RBP transcripts that show dysregulated expression in PSC and CCA is provided. Moreover, by utilizing a bioinformatic data mining approach, we give insight into those RBP transcripts that also exhibit differential expression in liver and gall bladder, as well as in body fluids, and are promising as biomarkers for diagnosing and predicting the prognosis of PSC. Expression data were bioinformatically extracted from public repositories usingTCGA Bile Duct Cancer dataset for CCA and specific NCBI GEO datasets for both PSC and CCA; more specifically, RBPs annotations were obtained from RBP World database. Interestingly, our comprehensive analysis shows an elevated expression of the non-canonical RBPs, FANCD2, as well as the microtubule dynamics regulator, ASPM, transcripts in the body fluids of patients with PSC and CCA compared with their respective controls, with the same trend in expression being observed in gall bladder and liver cancer tissues. Consequently, the manipulation of tissue expression of RBP transcripts might be considered as a strategy to mitigate the onset of CCA in PSC patients, and warrants further experimental investigation. The analysis performed herein may be helpful in the identification of non-invasive biomarkers for the early detection of PSC and for predicting its progression into CCA. In conclusion, future clinical research should investigate in more depth the full potential of RBP transcripts as biomarkers for human pathologies.

Chronic Liver Diseases: What is Up?

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Extracellular Vesicles as Delivery Vehicles for Non-Coding RNAs: Potential Biomarkers for Chronic Liver Diseases.

In recent years, EVs have emerged as promising vehicles for coding and non-coding RNAs (ncRNAs), which have demonstrated remarkable potential as biomarkers for various diseases, including chronic liver diseases (CLDs). EVs are small, membrane-bound particles released by cells, carrying an arsenal of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and other ncRNA species, such as piRNAs, circRNAs, and tsRNAs. These ncRNAs act as key regulators of gene expression, splicing, and translation, providing a comprehensive molecular snapshot of the cells of origin. The non-invasive nature of EV sampling, typically via blood or serum collection, makes them highly attractive candidates for clinical biomarker applications. Moreover, EV-encapsulated ncRNAs offer unique advantages over traditional cell-free ncRNAs due to their enhanced stability within the EVs, hence allowing for their detection in circulation for extended periods and enabling more sensitive and reliable biomarker measurements. Numerous studies have investigated the potential of EV-enclosed ncRNAs as biomarkers for CLD. MiRNAs, in particular, have gained significant attention due to their ability to rapidly respond to changes in cellular stress and inflammation, hallmarks of CLD pathogenesis. Elevated levels of specific miRNAs have been consistently associated with various CLD subtypes, including metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), and chronic hepatitis B and C. LncRNAs have also emerged as promising biomarkers for CLD. These transcripts are involved in a wide range of cellular processes, including liver regeneration, fibrosis, and cancer progression. Studies have shown that lncRNA expression profiles can distinguish between different CLD subtypes, providing valuable insights into disease progression and therapeutic response. Promising EV-enclosed ncRNA biomarkers for CLD included miR-122 (elevated levels of miR-122 are associated with MASLD progression and liver fibrosis), miR-21 (increased expression of miR-21 is linked to liver inflammation and fibrosis in CLD patients), miR-192 (elevated levels of miR-192 are associated with more advanced stages of CLD, including cirrhosis and HCC), LncRNA HOTAIR (increased HOTAIR expression is associated with MASLD progression and MASH development), and LncRNA H19 (dysregulation of H19 expression is linked to liver fibrosis and HCC progression). In the present review, we focus on the EV-enclosed ncRNAs as promising tools for the diagnosis and monitoring of CLD of various etiologies.

FLVCR1a Controls Cellular Cholesterol Levels through the Regulation of Heme Biosynthesis and Tricarboxylic Acid Cycle Flux in Endothelial Cells.

Feline leukemia virus C receptor 1a (FLVCR1a), initially identified as a retroviral receptor and localized on the plasma membrane, has emerged as a crucial regulator of heme homeostasis. Functioning as a positive regulator of δ-aminolevulinic acid synthase 1 (ALAS1), the rate-limiting enzyme in the heme biosynthetic pathway, FLVCR1a influences TCA cycle cataplerosis, thus impacting TCA flux and interconnected metabolic pathways. This study reveals an unexplored link between FLVCR1a, heme synthesis, and cholesterol production in endothelial cells. Using cellular models with manipulated FLVCR1a expression and inducible endothelial-specific Flvcr1a-null mice, we demonstrate that FLVCR1a-mediated control of heme synthesis regulates citrate availability for cholesterol synthesis, thereby influencing cellular cholesterol levels. Moreover, alterations in FLVCR1a expression affect membrane cholesterol content and fluidity, supporting a role for FLVCR1a in the intricate regulation of processes crucial for vascular development and endothelial function. Our results underscore FLVCR1a as a positive regulator of heme synthesis, emphasizing its integration with metabolic pathways involved in cellular energy metabolism. Furthermore, this study suggests that the dysregulation of heme metabolism may have implications for modulating lipid metabolism. We discuss these findings in the context of FLVCR1a's potential heme-independent function as a choline importer, introducing additional complexity to the interplay between heme and lipid metabolism.

COVID-19 and the enteric system: rapidly propagating issues.

The newly described SARS-CoV-2 respiratory virus is now righteously presenting as an ominous threat, based on the speed with which it originated a zoonosis from bats; advancing at a similar rate, the virus has placed mankind before a pandemic, with an infection toll of some 431 million, and a lethality of 5,9 million (as of February 25, 2022). The size of the harm that this agent can unleash against us is appallingly wide, from brain ischemia to foot chilblain, passing by heart massive infarction. Designing a possible response, we reappraised the well-known equation depression-inflammation, and tested the hypothesis that an upgraded ease-of-mind might help reduce the host's hospitality towards SARS-CoV-2. With time passing, it becomes increasingly evident that the virus shall tend to progressively occupy spaces, replacing pandemics with an apparently calm endemicity. This will have to be avoided, and surveillance of society on psychological terms will be one tenet. Needless to say, the role of the enteric tract in these issues is growing higher, and it will be narrated to seal the matters with the last (not the least) touch of glue.